Peptides for active anti-cytokine immunization

ABSTRACT

Peptide of a size comprised between 5 and 40 amino acids, originating from a cytokine, in which at least one of its amino acids comprises at least one of its atoms separated by a distance d of less than 5 angströms from an atom of the receptor corresponding to said cytokine, the spacing d being evaluated on the basis of structural data, derivatives, immunogenic compounds comprising them, use of a peptide or peptide derivative or immunogenic compound for the preparation of a curative or preventative medicament intended for the treatment or prevention of diseases linked to an excess or to the presence of cytokines or for the treatment of an auto-immune disease and pharmaceutical compositions which contain at least one abovementioned peptide or peptide derivative or immunogenic compound as active ingredient.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No.13/362,234, filed Jan. 31, 2012, which is a divisional of applicationSer. No. 13/004,588, filed Jan. 1, 2011, now issued as U.S. Pat. No.8,147,819, which is a divisional of application Ser. No. 12/628,826,filed Dec. 1, 2009, now issued as U.S. Pat. No. 7,892,529, which is adivisional of application Ser. No. 12/134,743, filed Jun. 6, 2008, nowissued as U.S. Pat. No. 7,641,895, which is a divisional of applicationSer. No. 10/510,116, now issued as U.S. Pat. No. 7,393,523, which is thenational stage under 35 U.S.C. 371 of PCT/FR2003/001120, filed Apr. 9,2003. The entire contents of prior application Ser. Nos. 10/510,116,12/134,743, 12/628,826, 13/004,588 and 13/362,234 are hereinincorporated by reference.

The present invention relates to new peptides and their use intherapeutics.

Active anti-cytokine immunization is an active immunotherapy strategydeveloped since 1990 by Zagury et al. which is based in particular onPatent Application WO 92/22577.

This idea was taken up by several scientific teams which have publishedin international scientific journals, active immunizations against theentire IFNα protein multimerized by treatment with glutaraldehyde(Gringeri et al., JAIDS 1999; 20:358-70), a chimeric TNFα proteinconsisting of coupling the native TNFα protein with a T epitope ofovalbumin (Dalum et al., Nature Biotechnology, 1999; 17:666-69), againstentire IL9 coupled with KLH (Richard et al., PNAS, 2000; 97:767-72) oralso chimeric entire IL5 with a T epitope of tetanus toxin (Hertz etal., J. Immunol, 2001; 167:3792-99).

These approaches have confirmed the feasibility of autologousanti-cytokine immunizations, but these few successes obscure theunsuccessful tests described by certain authors: certain cytokines donot allow sufficiently protective and clinically effective antibodies tobe obtained, and the same cytokine prepared in a form which is effectivein one manner, will not be effective in another (Richard et al., PNAS,2000; 97:767-72).

In trying to explain this phenomenon, the Applicant has observed that todate all the authors have used entire cytokines (optionally slightlymodified), which leads to difficulties in particular at the followinglevels:

dilution of the immunogenic power of the antigenic determinants ofinterest (for the B and T responses)

possible genesis of facilitating antibodies in vivo (B response).

It would therefore be desirable to have antigens available which make itpossible to obtain antibodies which are sufficiently protectivevis-à-vis cytokines, and limiting their activity.

WO-A-98/51705 describes h RANTES, MIP1α and MIP1β peptides comprisedbetween the second and third cysteine of these chemokines which bind theCCR5 co-receptor.

WO-A-98/34631 describes peptides originating from γ chains of cytokinereceptors used in order to block the binding of the cytokine oractivation of the receptor.

WO-A-94/01457 describes IFN α peptides used as substances carryingpharmaceutical compounds.

EP-A-0218531 describes IL1 peptides used for the preparation ofantibodies.

This is why a subject of the present application is peptides of a sizecomprised between 5 and 40 amino acids, originating from a cytokine,characterized in that at least one of its amino acids and preferably atleast two of its consecutive amino acids comprise at least one of itsatoms separated by a distance d of less than 5 angströms from an atom ofthe receptor corresponding to said cytokine, the spacing d beingevaluated on the basis of structural data (for example crystallographyor NMR) and preferably in that they induce antibodies interacting withsaid cytokine, with the exception

-   -   of the peptides comprised between the 2nd and 3rd cysteine of h        RANTES, MIP 1α and MIP 1β, and    -   of the peptides comprised between amino acids 123 and 140 of IFN        α.

Because of their length, the cytokine peptides according to theinvention correspond to a limited number of cytokine epitopes, ingeneral to one or two cytokine epitopes, and are consequently depletedof a number of other epitopes that they contain.

By “cytokine”, is meant both true cytokines as well as chemo-attractivecytokines also called chemokines. Human cytokines are preferred. By“interacting” is meant that these antibodies, for example either becausethey recognize the native protein, as can be shown by an immunologicaltest (ELISA, Western Blot), or because they block the binding of thecytokine to its receptor as can be shown by a biological activity testor a biochemical competition test, have a beneficial clinical effect invivo.

Amongst the cytokines, TGF β, IL1 α, IL1 β, vEGF, TNF α, IFN α and γ, IL4, 5, 6, 10, 12, 13, 15, 18, 23, IP10, MIP 1α and 1β, MCP1, and Rantescan for example be mentioned. Amongst the above cytokines, TGF β, IL1 β,vEGF, TNF α, IFN α and γ, IL 4, 5, 6, 10, 12, 13, 15, 23 are preferred,or any combination of certain of these cytokines and quite particularlyIL1 β, vEGF, TNF α, IL23 and IFN α or any combination of certain ofthese cytokines.

The cytokine peptides according to the invention originate or derivefrom a cytokine. By “originate” is meant that their amino acid sequenceis identical to that of the cytokine. By “derive” is meant that theiramino acid sequence is mostly identical to that of the cytokine butcomprises a few differences as will be seen hereafter.

At least one amino acid of the cytokine peptides of the invention andpreferably two consecutive amino acids possess(es) one of its atomsseparated by less than 5 angströms from an atom of the receptorcorresponding to said cytokine. It is advantageously separated by lessthan 4.5 angströms, in particular separated by less than 4 angströms andparticularly separated by less than 3.5 angströms from an atom of thereceptor corresponding to said cytokine.

In general the atom concerned of the amino acid is situated on the sidechain of said amino acid.

Under preferential conditions for implementation of the invention 2, inparticular 3 and preferably 4 consecutive amino acids of the cytokinepeptide correspond to this same spacing criterion.

This spacing is evaluated on the basis of structural data, for exampleof crystallography, or also by NMR which produces results similar to thecrystallographic measurements.

The above cytokine peptides advantageously comprise more than 8, inparticular more than 10, particularly more than 12 and quiteparticularly more than 15 amino acids.

Under other preferential conditions of implementation of the invention,the above cytokine peptides comprise less than 35, advantageously lessthan 30, in particular less than 25 and particularly less than 20 aminoacids. Generally the shortest sequences correspond to peptidescontaining a single group of at least two consecutive amino acidscomprising at least one of their atoms separated by less than 5angströms from an atom of the receptor corresponding to said cytokine,whereas the longest sequences correspond in general to peptidesaccording to the invention containing two or even three groups or moreof such consecutive amino acids. In fact these groups can be separatedby several, for example 10 amino acids, as in the case of IL1β.

From the above cytokine peptides, one or more peptides are in particularretained, chosen from or originating from those, the names of whichfollow:

hIL1β (Human Interleukin 1 beta) (SEQ ID No. 1) 1-APVRSLNCTL-10(SEQ ID No. 2) 29-LHLQGQDMEQQ-39 (SEQ ID No. 3) 123-STSQAENMPV-132hvEGF (Human vascular Endothelial Growth Factor) (SEQ ID No. 4)73-IMRIKPHQGQHIGEMS-88 hTNFα (Human Tumor Necrosis Factor alpha)(SEQ ID No. 5) 20-PQAEGQLQWLNRRANALLANGVELRDNQLVVPSEG-54 (SEQ ID No. 6)80-ISRIAVSYQTKVNLLS-95 (SEQ ID No. 7) 124-FQLEKGDRLSAEINR-138 hIFNγ(Human Interferon gamma) (SEQ ID No. 8)1-MQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKN-36 (SEQ ID No. 9)118-MAELSPAAKTGKRKRS-133 hIL10 (Human Interleukin 10) (SEQ ID No. 10)20-PNMLRDLRDAFSRVKTFFQMKDQLDNLLLKE-50 hIL4 (Human Interleukin 4)(SEQ ID No. 11) 5-ITLQEIIKTLNSL-17 (SEQ ID No. 12)70-AQQFHRHKQLIRFLKRLDRNLWGLAG-95hIL12p40 (Human Interleukin 12 sub-unit p40) (SEQ ID No. 13)80-LLLHKKEDGIWSTDILKDQKEPKNKTFLRCE-110 (SEQ ID No. 14)135-KSSRGSSDPQG-145 hIL18 (Human Interleukin 18) (SEQ ID No. 15)1-YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTD-35 (SEQ ID No. 16)68-CEKISTLSCEN-78 (SEQ ID No. 17) 141-EDELGDRSIMFTVQNED-157hIP10 (Human Interferon gamma inducible protein) (SEQ ID No. 18)39-VEIIATMKKKGEKRCLNPESKA-60 hIL5 (Human Interleukin 5) (SEQ ID No. 19)1-IPTSALVKETLALLSTHRTLLIANET-26 (SEQ ID No. 20) 96-LQEFLGVMNTEWI-108hTGFI32 (Human Transforming Growth Factor beta type 2) (SEQ ID No. 21)25-KRDLGWKWIHE-35 (SEQ ID No. 22) 87-TILYYIGKTPKIEQ-100hIL15 (Human Interleukin 15) (SEQ ID No. 23) 1-ANWVNVISDLKKI-13(SEQ ID No. 24) 74-SSNGNVTESGCKECEELEKKNIKEFLQSFVHIVQMF-111hIL6 (Human Interleukin 6) (SEQ ID No. 25) 28-KQIRYILDGISA-39(SEQ ID No. 26) 114-RAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLL-149 hMIP1α(Human Macrophage Inflammatory Protein alpha) (SEQ ID No. 27)51-ADPSEEWVQKYVSDLELSA-69 hMIP1β (Human Macrophage InflammatoryProtein beta) (SEQ ID No. 28) 52-ADPSESWVQEYVYDLELN-69hIL13 (Human Interleukin 13) (SEQ ID No. 29) 8-TALRELIEEL-17(SEQ ID No. 30) 57-CSAIEKTQRMLSGFCPHKVSAGQFSS-82hIL23 (Human Interleukin 23) (SEQ ID No. 31) 52 GHMDLREEGDEETT 65(SEQ ID No. 32) 115 LLPDSPVGQLHASLLGLSQ 133 (SEQ ID No. 33)160 LLRFKILRSLQAFVAVAARV 179hRANTES (Human Regulated upon Activation Normal T-cell expressed)(SEQ ID No. 34) 51-ANPEKKWVREYINSLEMS-68 hIFNα (Human Interferon alpha)(SEQ ID No. 35) 12-RRTLMLLAQMRK-23 (SEQ ID No. 36)95-LEACVIQGVGVTETPLMKEDSILAVRK-121

The preceding sequences were chosen from the cytokines which participatein the development of human diseases due to their role in theinflammatory response or specific immune response.

The following peptides are quite particularly retained:

(SEQ ID No. 3) 123-STSQAENMPV-132 of hIL1β (SEQ ID No. 4)73-IMRIKPHQGQHIGEMS of hvEGF (SEQ ID No. 5)20-PQAEGQLQWLNRRANALLANGVELRDNQLVVPSEG-54 of hTNFα (SEQ ID No. 8)1-MQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKN-36 of hIFNγ (SEQ ID No. 9)20-PNMLRDLRDAFSRVKTFFQMKDQLDNLLLKE-50 of hIL10 (SEQ ID No. 11)70-AQQFHRHKQLIRFLKRLDRNLWGLAG-95 of hIL4 (SEQ ID No. 15)1-YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTD-35 of hIL18 (SEQ ID No. 31)52 GHMDLREEGDEETT 65, (SEQ ID No. 32) 115 LLPDSPVGQLHASLLGLSQ 133 or(SEQ ID No. 33) 160 LLRFKILRSLQAFVAVAARV 179 of hIL23.

As is known by the person skilled in the art of immunology,modifications of the natural peptide chains are possible without howevermodifying the nature of the immunological properties of the immunogenicpeptides. Derivatives of cytokine peptides can therefore also bementioned, which are highly homologous to these natural sequences, forexample having more than 50% homology, in particular more than 70%homology, and preferably more than 80% homology or even more than 90%homology with the corresponding native peptide whilst retaining theimmunological properties of this epitopic site of the native peptide.Their homology zone can vary from 5 to 40 residues, for example from 8to 40 residues, or also from 8 to 35 residues, preferably from 10 to 35residues but also from 12 to 35 residues, notably from 12 to 30residues, in particular from 15 to 30 residues and quite particularlyfrom 15 to 25 residues.

The cytokine peptide derivatives can contain modified residues, oncondition that the modifications do not appreciably reduce theimmunogenicity, either by adding chemical radicals (methyl, acetyl etc.)or by stereochemical modification (use of D series amino acids). Thecytokine peptide derivatives should, like the cytokine peptides induceantibodies interacting with cytokine.

The cytokine peptide derivatives according to the invention can compriseone or more modifications in the amino acids of which they areconstituted, such as deletions, substitutions, additions, orfunctionalizations (such as acylation) of one or more amino acids, tothe extent that these modifications remain within the frameworkspecified above (immunological characters). For example, in general thereplacement of a leucine residue by an isoleucine residue does notmodify such properties; the modifications should generally concern lessthan 40% of the amino acids, in particular less than 30%, preferablyless than 20% and quite particularly less then 10% of the amino acids ofthe natural peptide. It is important that the antibodies induced by themodified peptides are active vis-à-vis native cytokine.

These modifications are within the scope of a person skilled in the art,who can verify the incidence of the modifications by simple tests. Theimmunogenicity of such modified derivatives can be evaluated by ELISAafter immunization of mice, the antigen tested by ELISA being the entirecytokine or the immunizing cytokine peptide, or by cytokine-receptorbond blocking tests. The possible modifications preferably affect lessthan 8 amino acids, advantageously less than 6 amino acids, inparticular less than 4 amino acids, and particularly 3 amino acids orless, such as 2 or 1 single amino acid.

A subject of the invention is also a compound characterized in that itcontains at least one abovementioned cytokine peptide or cytokinepeptide derivative. Such a compound can comprise identicalpeptide/derivative repetitions, or different peptide/derivativecombinations, either in linear form or in the form of a candelabrastructure or couplings mixed with carrier proteins. Such a compound canalso be presented in cyclized form. Thus cytokine peptides or cytokinepeptide derivatives according to the invention can for example beinserted into longer sequences of amino acids providing in particular abetter conformation or combined with exogenous T epitopes (whether forprotein or DNA immunizations).

They can advantageously be associated in a covalent manner with carrierproteins such as for example KLH.

As was seen, the cytokine peptides according to the invention correspondin general to a small number of cytokine epitopes. When they are inparticular inserted, combined or associated, the above compounds do notcomprise other epitopes of said cytokine.

These cytokine peptides or cytokine derivatives can be included in anyprotein sequence which comprises no homology with the other epitopes ofthe natural cytokine. For example, they can be sites binding thereceptor, to the ends of which a cysteine is simply added in order toconfer a cyclic structure on the peptide. Another example is a peptidesurrounded by sequences of T epitopes of the tetanus toxin. Yet anotherexample can comprise a peptide corresponding to the sequence of thereceptor binding site but where certain amino acids are replaced bytheir D series isomers in order to avoid their agonist effect. In fact,it can be optionally advantageous to use peptide derivatives which haveno agonist activity on the receptor so that the immunogen does notinterfere with the immune response.

In order to increase the immune response, these cytokine peptides orcytokine derivatives can be coupled to carrier proteins. The couplingmethods and the carrier protein considered can be different according tothe target peptide: they can for example be Keyhole Limpet Hemocyanin(KLH) protein and Tetanus Toxoid (TT) conjugated to the peptides bychemical methods well known to a person skilled in the art such as thoseof carbodiimide, glutaraldehyde or bis-diazotized benzidine coupling.The realization of these couplings can be facilitated by the addition orincorporation of amino acids into the sequence, such as for examplelysines, histidines, tyrosines or cysteines. Such peptide compoundscoupled to an exogenous T epitope (originating from plasmodiumfalciparum, KLH, etc.) whether chemically or genetically also comewithin the scope of the invention.

Network couplings of candelabra type or to molecules such as transferrinor ferritin can also be implemented in order to effectively stimulatethe immune response.

The peptides according to the invention can in particular be produced bychemical synthesis or genetic engineering or any other suitable method.The synthesis of cyclic peptides, grafting, as needed, one or more aminoacids at the end of the chain as cysteines in order to create adisulphide bridge makes it possible to recover part of the secondarystructure that these peptide fragments possess in the three-dimensionalstructure of the protein.

The cytokine peptides, cytokine derivatives and their compoundsaccording to the invention possess very useful pharmacologicalproperties. They are in particular endowed with remarkable anti-cytokineproperties. They are in fact immunogens and capable of generating in asubject antibodies recognizing the native cytokine. These peptides donot contain the numerous other epitopes originating from the cytokine towhich they correspond.

These properties are illustrated hereafter in the experimental part.They justify the use of the peptides described above as medicaments.

The fact of being limited to these peptides close to the receptorbinding site, to the exclusion of the other cytokine epitopes, inparticular provides the advantage of limiting the generation ofantibodies facilitating or potentializing cytokine activity. Moreover,they make it possible to increase the quality of the anticytokineimmunization as the number of target antigenic determinants is limited.

This is why a subject of the invention is also medicaments characterizedin that they are constituted by cytokine peptides or cytokinederivatives or compounds as defined above, i.e. cytokine peptides orcytokine derivatives or immunogenic compounds as defined above, orcomprised between the 2nd and 3rd cysteine of h RANTES or comprisedbetween amino acids 123 and 140 of IFN α, for their use in a method oftherapeutic treatment of the human or animal body, as well as the use ofsuch a cytokine peptide or cytokine derivative or immunogenic compoundfor the preparation of a curative or preventative medicament intendedfor the treatment or prevention of diseases linked to an excess or tothe presence of cytokines.

The medicaments according to the present invention are used for examplein both the curative and preventative treatment of the diseases linkedto deregulation of the immune system involving overproduction ofcytokines such as for example auto-immunes diseases (including, interalia, multiple sclerosis, rheumatoid polyarthritis, psoriasis,auto-immune diabetes, lupus), allergy or asthma, cancers or AIDS. Thusit is clear that combating IL1β or TNFα can be useful in rheumatoidpolyarthritis, combating IFNγ, IL18, IL23 or IL12 can be useful againstmultiple sclerosis or auto-immune diabetes, combating IL4, IL5 and IL13can be useful against allergy or asthma, combating IL10 or vEGF can beuseful against certain cancers. More generally the Th1/Th2 deregulationswhich govern the immune response itself and which in a standard fashioncause IL12, IL2, IL4, IL6, IL10, IL13, IFNγ, TNFα to intervene, canbenefit from a readjustment of the equilibrium by active immunization.These are only a few examples, and a subject of the invention is alsoany treatment of the human body, based on active immunization (DNA orpeptide) involving the abovementioned peptide sequences to the exclusionof the other cytokine epitopes. These sequences can be modified asindicated in the present description, and the DNA immunizations arecarried out by simple translation from the genetic code.

The humoral immune response can be evaluated by ELISA tests or testsshowing the inhibition of the native cytokine bond to its receptor. Thecell response can be evaluated in the presence of cell proliferationtests vis-à-vis the peptide used.

The immunogenic active ingredients according to the invention can beused as follows:

A cytokine peptide or cytokine derivative or immunogenic compoundaccording to the present invention, is administered to a patient, forexample by sub-cutaneous or intramuscular route, in a sufficientquantity to be effective at a therapeutic level, to a subject needingsuch treatment. The dose administered can for example range from 1 to1000 μg, in particular 10 to 500 μg, by sub-cutaneous route, once amonth for three months, then periodically as a function of the inducedserum antibodies count, for example every 2-6 months. In the samepreparation two or more different immunogenic molecules can beadministered in order to induce antibodies neutralizing all thedeleterious functional sites in the case where a single immunogenicmolecule does not carry all the active sites of the overproducedcytokine that are to be neutralized.

A subject of the invention is also the pharmaceutical compositions inparticular the vaccines which contain at least one abovementionedcytokine peptide or cytokine derivative or immunogenic compound, asactive ingredient.

As medicaments, a cytokine peptide or cytokine derivative or immunogeniccompound of the invention can be incorporated into pharmaceuticalcompositions intended for any standard route in use in the field ofvaccines, in particular by sub-cutaneous route, by intramuscular route,by intravenous route or by oral route. The administration can take placein a single dose or repeated once or more after a certain period oftime.

This is why a subject of the present application is also a curative orpreventative pharmaceutical composition, characterized in that itcomprises as active ingredient, one or more cytokine peptides orcytokine derivatives or compounds immunogenes which are new or comprisedbetween amino acids 123 and 140 of IFN α, as defined above.

The immunogenic agent can be conditioned alone or mixed with anexcipient or mixture of pharmaceutically acceptable excipients as anadjuvant. A subject of the present application is more particularly avaccine containing as immunogen, an abovementioned cytokine peptide orcytokine derivative or immunogenic compound.

A subject of the present invention is also a process for preparing acomposition described above, characterized in that, according to methodsknown per se, the active ingredient or ingredients are mixed withacceptable, in particular pharmaceutically acceptable excipients.

The administration to a patient of a cytokine peptide or cytokinederivative or immunogenic compound according to the inventioncorresponds to an active immunotherapy. It can also be useful to carryout passive immunotherapy, i.e. to provide a patient directly with theantibodies which he needs.

The vaccine preparations can be packaged for the intra-nasal route inthe form of gel with carbopol as excipient, of nasal drops or spray andfor the oral route in the form of gastroresistant capsules, sugar-coatedtablets or gastroresistant granules.

In the case of DNA vaccine administered by systemic or mucosal route,the galenic presentation of the plasmid can be a suspension in aphysiological liquid such as physiological PBS (phosphate bufferedsaline=PBS). The plasmids can be enclosed in biodegradable polymer (PLG,PLA, PCL) microspheres and administered in gastroresistant capsules foringestion (oral route). The DNA can also be expressed in a bacterial,salmonella-type or viral-type, adenovirus or poxvirus living vector.

Finally, a subject of the present application is a process for activeimmunization of patients characterized in that as immunogen, a cytokinepeptide or cytokine derivative or immunogenic compound is used, asdefined above, advantageously associated with a mineral, oily orsynthetic immunity adjuvant.

The immunizations can be done in a standard fashion in particular bypeptides or immunogenic compounds as conjugates preferably in thepresence of an adjuvant, for example ISA 51 or Alum. The immunizationscan be DNA-based (sequences homologous to the binding sites combinedwith exogenous T epitopes) using naked DNA or an expression vectorcontaining an adapted promoter such as for example pCR3.1. The DNAsadministered can be protected from the nucleases by the use ofappropriate radicals (CpG etc.). In particular an initial DNAimmunization can be followed by standard boosters using the peptidecompounds.

The preferential conditions for implementation of the peptides describedabove also apply to the other subjects of the invention referred toabove.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows the crystallographic structure of the peptideoriginating from IL10 in contact with its receptor. The IL10 cytokine(on the left) can be seen in contact with its receptor (on the right).

FIG. 2 is an enlargement of a portion of FIG. 1.

In FIG. 2, it can be observed that the amino acids marked in black areseparated by less than 4 Angströms (d=dotted line between two blackdots). They correspond, from bottom to top, to the pairs: 192IL10Rserine vis-à-vis 28IL10 aspartic acid (3.18 Å), 100IL10R aspartic acidand 101IL10R glutamic acid vis-à-vis 34IL10 lysine (3.83 Å and 3.84 Å),and 94IL10R asparagine vis-à-vis 39IL10 methionine (3.70 Å). For eachamino acid a central point was determined as being the baric centre ofthe alpha carbon of the amino acid considered, the end of the side chainand a third point chosen as being the furthest from the preceding two,on the side chain. The distance d measured is the distance separatingthe central points of the amino acids, respectively of the cytokine andits receptor. The distances can evidently be defined using other usualmethods.

The following examples illustrate the present invention.

EXAMPLE 1

The peptide KLHLQGQDMEQQ (SEQ ID No. 37) was synthesized from thesequence of human IL1β, the K residue was added to the natural sequencein order to allow binding with KLH by coupling using glutaraldehyde.Five mice were immunized in the presence of ISA51 adjuvant. For thefirst injection carried out on day 0, an emulsion is prepared of 40 μgof immunogen in a quantity of ISA51 sufficient for preparing 100 μl ofemulsion. For the booster on days 21 and 40, 100 μL of emulsion isprepared containing 20 μg of immunogen. Five mice were immunized inparallel as controls, with KLH in the same adjuvant.

On day 60, serum was taken and ELISA tests carried out with the nativecytokine in order to reveal the presence of antibodies.

The average absorbance obtained for each mouse is presented in the tablebelow:

Mouse 1 2 3 4 5 Average Peptide 1.34 0.08 1.86 0.90 1.56 1.15 Control0.05 0.11 0.08 0.12 0.06 0.08

The existence of antibodies neutralizing the native cytokine wasevaluated as follows: 20 units of native IL1β cytokine are preincubatedwith serum from mice (immunized or control) diluted from 1/100^(th) to1/2000^(th) over 2 hours at 37° C. The whole is then added to EL4 cells,with 50,000 cells per microwell. The production of IL2 is evaluatedafter 24 hours in the supernatant by a sandwich ELISA test (R&Ddiagnostics). The percentage inhibition of the production of IL2indicates the percentage of neutralization of the IL1β. As can be seen,responses are observed for the immunized mice, but not for the controls.

Mouse 1 2 3 4 5 Average Peptide 52% 25% 93% 86% 8% 52% Control 8% −5%11% 5% 12% 6%

EXAMPLE 2

5 mice were immunized with the synthetic peptide originating from humanvEGF: KPHQGQHIGEMS. (SEQ ID No. 38) This peptide was coupled to thecarrier protein KLH by reaction with glutaraldehyde. After immunizationunder the same conditions as in Example 1, serum is taken on day 60 andELISA tests are carried out with the native cytokine in order to revealthe presence of antibodies.

The average absorbance obtained for each mouse is presented in the tablebelow:

Mouse 1 2 3 4 5 Average Peptide 1.50 1.00 0.86 0.98 1.63 1.19 Control0.05 0.13 0.07 0.10 0.08 0.09

EXAMPLE 3

5 mice were immunized with the synthetic peptide originating from humanTNFα which is: KYQAEGQLQWLNRRANALLANGVELRDNQL. (SEQ ID No. 39) Thispeptide was coupled to the carrier protein KLH by reaction withdiazobenzidine. The K and Y residues were added to the natural sequencein order to allow binding with the KLH by coupling using glutaraldehyde.After immunization under the same conditions as in Example 1, serum istaken on day 60 and ELISA tests are carried out with the native cytokinein order to reveal the presence of antibodies.

The average absorbance obtained for each mouse is presented in the tablebelow:

Mouse 1 2 3 4 5 Average Peptide 1.64 1.14 1.96 1.02 0.78 1.31 Control0.11 0.16 0.09 0.11 0.18 0.13

The existence of antibodies neutralizing the native cytokine wasevaluated as follows: 50 units of native TNFα cytokine are preincubatedwith serum from mice (immunized or control) diluted 1/200^(th) over 2hours at 37° C. The whole is then added to L929 cells, with 25,000 cellsper microwell. The lysis of the cells is evaluated after 24 hours bystaining with naphthol blue black (Sigma). The percentage inhibition ofthe lysis indicates the percentage of neutralization of the TNFα, and ascan be seen, responses are observed for the immunized mice but not thecontrols.

Mouse 1 2 3 4 5 Average Peptide 88% 12% 93% 36% 79% 61% Control 5% 6%14% 5% 1% 6%

EXAMPLE 4

5 mice were immunized with the peptide originating from human IFNγ whichis: KKYFNAGHSDVADNGTLFLGILKN (SEQ ID No. 40). This peptide wassynthetized chemically in the form of MAPS. This MAPS peptide was thencoupled to the carrier protein Tetanus Toxoid by reaction withglutaraldehyde. After immunization under the same conditions as inExample 1, serum is taken on day 60 and ELISA tests are carried out withthe native cytokine in order to reveal the presence of antibodies.

The average absorbance obtained for each mouse is presented in the tablebelow:

Mouse 1 2 3 4 5 Average Peptide 1.56 1.05 0.98 1.80 1.64 1.41 Control0.10 0.12 0.23 0.08 0.09 0.12

The existence of antibodies neutralizing the native cytokine wasevaluated as follows: 100 units of native cytokine are pre-incubatedwith serum from mice (immunized or control) diluted to 1/250^(th) over 2hours at 37° C. The whole is then added to RAW 264.7 cells, with 300,000cells being placed in 2 ml wells. The expression of class II CMH on thecells is evaluated by flux cytometry after 24 hours, by marking withclass II anti-CMH antibodies coupled with fluorescein. The percentageinhibition of the expression of the class II CMH indicates thepercentage of neutralization of the IFNγ, and as can be seen,inhibitions are observed for the immunized mice but not the controls.The significance threshold for these experiments is 30%.

Mouse 1 2 3 4 5 Average Peptide 40% 89% 67% 38% 83% 63% Control 25% 14%−10% 29% 7% 13%

EXAMPLE 5

5 mice were immunized with the peptide originating from human IL10 whichis: DECNMLRDLRDAFSRVKTFFQMKDQLDNC (SEQ ID No. 41). This peptide wassynthesized chemically, and a cysteine was added to each end in order tomake a cyclic peptide. This peptide was then coupled with the carrierprotein KLH by reaction with carbodiimide. The DE residue was also addedto the natural sequence in order to allow binding with the KLH byreaction with carbodiimide. After immunization under the same conditionsas in Example 1, serum is taken on day 60 and ELISA tests are carriedout with the native cytokine in order to reveal the presence ofantibodies.

The average absorbance obtained for each mouse is presented in the tablebelow:

Mouse 1 2 3 4 5 Average Peptide 1.94 0.96 1.85 0.97 1.5 1.44 Control0.29 0.52 0.17 0.26 0.36 0.32

EXAMPLE 6

5 mice were immunized with the peptide corresponding to human IL4 whichis: KQLIRFLKRLDRNLWGLAG (SEQ ID No. 42). This peptide was coupled withthe carrier protein KLH by reaction with glutaraldehyde. Afterimmunization under the same conditions as in Example 1, serum is takenon day 60 and ELISA tests are carried out with the native cytokine inorder to reveal the presence of antibodies.

The average absorbance obtained for each mouse is presented in the tablebelow:

Mouse 1 2 3 4 5 Average Peptide 1.06 1.25 0.95 0.87 1.24 1.07 Control0.34 0.09 0.26 0.16 0.12 0.19

EXAMPLE 7

5 mice were immunized with the peptide corresponding to human IL12p40which is: KKEDGIWSTDILKDQKEPKNKTFLRCE (SEQ ID No. 43). This peptide wascoupled with the carrier protein Tetanus Toxoid by reaction withbis-diazotized benzidine (diazo). After immunization under the sameconditions as in Example 1, serum is taken on day 60 and ELISA tests arecarried out with the native cytokine in order to reveal the presence ofantibodies.

The average absorbance obtained for each mouse is presented in the tablebelow:

Mouse 1 2 3 4 5 Average Peptide 0.95 1.06 1.58 1.14 0.87 1.12 Control0.06 0.12 0.09 0.05 0.09 0.08

EXAMPLE 8

5 mice were immunized with the peptide originating from human IL18 whichis: KYFGKLESKLSVIRNLNDQVLFID (SEQ ID No. 44). This peptide was coupledwith the carrier protein KLH by reaction with glutaraldehyde. The Kresidue was added to the natural sequence in order to allow binding withthe KLH by coupling using glutaraldehyde. After immunization under thesame conditions as in Example 1, serum is taken on day 60 and ELISAtests are carried out with the native cytokine in order to reveal thepresence of antibodies.

The average absorbance obtained for each mouse is presented in the tablebelow:

Mouse 1 2 3 4 5 Average Peptide 1.85 1.13 0.99 1.24 1.54 1.35 Control0.31 0.09 0.27 0.16 0.24 0.21

EXAMPLE 9

5 mice were immunized with the synthetic peptide originating from humanIP10 which is: KKKGEKRCLNPESKA (SEQ ID No. 45). This peptide was coupledwith the carrier protein Tetanus Toxoid by reaction with glutaraldehyde.The three K residues present in the natural sequence allow binding withthe KLH by coupling using glutaraldehyde. After immunization under thesame conditions as in Example 1, serum is taken on day 60 and ELISAtests are carried out with the native cytokine in order to reveal thepresence of antibodies.

The average absorbance obtained for each mouse is presented in the tablebelow:

Mouse 1 2 3 4 5 Average Peptide 1.16 0.98 1.24 1.09 0.88 1.07 Control0.32 0.21 0.11 0.20 0.09 0.19

EXAMPLE 10

5 mice were immunized with the peptide corresponding to human IL5 whichis: KLQEFLGVMNTEWI (SEQ ID No. 46). This peptide was coupled with thecarrier protein KLH by reaction with glutaraldehyde. The K residue wasadded to the natural sequence in order to allow binding with the KLH bycoupling using glutaraldehyde. After immunization under the sameconditions as in Example 1, serum is taken on day 60 and ELISA tests arecarried out with the native cytokine in order to reveal the presence ofantibodies.

The average absorbance obtained for each mouse is presented in the tablebelow:

Mouse 1 2 3 4 5 Average Peptide 1.25 1.21 1.87 1.10 0.88 1.26 Control0.51 0.21 0.42 0.09 0.38 0.32

EXAMPLE 11

5 mice were immunized with the peptide corresponding to human TGFβ2which is: DTILYYIGKTPKIE (SEQ ID No. 47). This peptide was coupled withthe carrier protein KLH by reaction with carbodiimide. The D residue wasadded to the natural sequence in order to produce a bond by coupling.After immunization under the same conditions as in Example 1, serum istaken on day 60 and ELISA tests are carried out with the native cytokinein order to reveal the presence of antibodies.

The average absorbance obtained for each mouse is presented in the tablebelow:

Mouse 1 2 3 4 5 Average Peptide 0.95 1.15 0.99 1.17 1.08 1.07 Control0.40 0.09 0.35 0.26 0.29 0.28

EXAMPLE 12

5 mice were immunized with the peptide corresponding to human IL6 whichis: KQIRYILDGISA (SEQ ID No. 25). This peptide was coupled with thecarrier protein TT by reaction with bis-diazotized benzidine (diazo).After immunization under the same conditions as in Example 1, serum istaken on day 60 and ELISA tests are carried out with the native cytokinein order to reveal the presence of antibodies.

The average absorbance obtained for each mouse is presented in the tablebelow:

Mouse 1 2 3 4 5 Average Peptide 1.11 1.27 1.02 1.34 0.32 1.01 Control0.11 0.15 0.29 0.14 0.21 0.21

EXAMPLE 13

The DNAs corresponding to the peptides 1-APVRSLNCTL-10(GCACCTGTACGATCACTGAACTGCACGCTC) (SEQ ID No. 48), 29-LHLQGQDMEQQ-39(CTCCACCTCCAGGGACAGGATATGGAGCAACAA) (SEQ ID No. 49) and123-STSQAENMPV-132 (AGCACCTCTCAAGCAGAAAACATGCCCGTC) (SEQ ID No 50) ofIL1β were synthesized and are separated from the T epitopes of thetetanus toxin:

(SEQ ID No. 55) AQYIKANSKFIGITEL (SEQ ID No. 51)(CAGTACATCAAGGCTAACTCCAAGTTCATCGGTATCACTGAGCTG) and the KLH: VDTWRKNVDSL(SEQ ID No. 52) (GTTGACACCACCAGAAAAAATGTTGACTCCCTT), (SEQ ID No. 54)by GYG spacers (GGCTACGGC).The final nucleotide sequence is as follows:

(SEQ ID No. 53) GTTGACACCACCAGAAAAAATGTTGACTCCCTTGGCTACGGCGCACCTGTACGATCACTGAACTGCACGCTCGGCTACGGCGTTGACACCACCAGAAAAAATGTTGACTCCCTTGGCTACGGCCTCCACCTCCAGGGACAGGATATGGAGCAACAAGGCTACGGCCAGTACATCAAGGCTAACTCCAAGTTCATCGGTATCACTGAGCTGGGCTACGGCAGCACCTCTCAAGCAGAAAACATGCCCGTCGGCTACGGCGTTGACACCACCAGAAAAAATGTTGACTCCCTT.

This DNA sequence, cloned in pRSET-A, therefore codes for a polypeptidecompound that is produced and purified by genetic engineering in theform of poly-Histidine fusion protein. This polypeptide is used asimmunogen as in Example 1.

The antibody response of the mice is measured against the native IL1β byELISA and the following values are found:

Mouse 1 2 3 4 5 Average Peptide 0.86 0.73 1.68 1.55 1.83 1.33 Control0.23 0.11 0.21 0.29 0.17 0.20

EXAMPLE 14

The cDNAs corresponding to the peptides 1-APVRSLNCTL-10(GCACCTGTACGATCACTGAACTGCACGCTC) (SEQ ID No. 48), 29-LHLQGQDMEQQ-39(CTCCACCTCCAGGGACAGGATATGGAGCAACAA) (SEQ ID No. 49) and123-STSQAENMPV-132 (AGCACCTCTCAAGCAGAAAACATGCCCGTC) (SEQ ID No. 50) ofIL1β were synthesized and separated from the T epitopes of the tetanustoxin

(SEQ ID No. 51) AQYIKANSKFIGITEL(CAGTACATCAAGGCTAACTCCAAGTTCATCGGTATCACTGAGCTG) and the KLH:(SEQ ID No. 52) VDTVVRKNVDSL (GTTGACACCACCAGAAAAAATGTTGACTCCCTT)by GYG spacers.The final sequence is as follows:

(SEQ ID No. 53) GTTGACACCACCAGAAAAAATGTTGACTCCCTTGGCTACGGCGCACCTGTACGATCACTGAACTGCACGCTCGGCTACGGCGTTGACACCACCAGAAAAAATGTTGACTCCCTTGGCTACGGCCTCCACCTCCAGGGACAGGATATGGAGCAACAAGGCTACGGCCAGTACATCAAGGCTAACTCCAAGTTCATCGGTATCACTGAGCTGGGCTACGGCAGCACCTCTCAAGCAGAAAACATGCCCGTCGGCTACGGCGTTGACACCACCAGAAAAAATGTTGACTCCCTT.This DNA sequence, cloned in pCR3.1, therefore codes for a IL1bpolypeptide under the control of the CMV promoter.

A first intra-muscular injection is carried out by injecting 100 μg ofvector in a saline solution of 100 μl. 2 boosters are carried out ondays 21 and 40 with the peptide of Example 13 (“prime-boost” typeimmunization). On day 60 the antibody response is evaluated by ELISAtest on the native cytokine on the 5 immunized mice and the 5 controlmice.

The result is as follows:

Mouse 1 2 3 4 5 Average Peptide 1.54 0.35 1.88 1.64 0.24 1.13 Control0.13 0.11 0.18 0.08 0.23 0.14

EXAMPLE 15

The vaccine is formed from a water-in-oil emulsion constituted by 50% ofISA 51 (Seppic, Paris) and 50% of an aqueous solution of the peptide ofExample 1 (50 μg/dose).

EXAMPLE 16 Plasmid-Immunogen-Based Vaccine for DNA Vaccination of IL10Systemic Type

Plasmids coding for the peptides APVRSLNCTL and LHLQGQDMEQQ of IL1β (20μg/dose) were suspended in 0.2 ml of PBS for intramuscularadministration.

EXAMPLE 17

A vaccine was prepared, formed from a water-in-oil emulsion constitutedby 50% of ISA (SEPPIC, Paris) and 50% of an aqueous solution of thesynthetic peptide KYQAEGQLQWLNRRANALLANGVELRDNQL derived from human TNFαcoupled with KLH (100 μg/dose).

1. An immunizing composition, comprising a pharmaceutically acceptableexcipient and, as active ingredient, an isolated peptide consisting of12 to 40 amino acid residues, said peptide originating from a humancytokine, wherein said isolated peptide comprises a sequence selectedfrom the group consisting of SEQ ID NO:27, SEQ ID NO:28, and SEQ IDNO:34.
 2. The immunizing composition of claim 1, wherein said peptidecomprises SEQ ID NO:27.
 3. The immunizing composition of claim 1,wherein said peptide comprises SEQ ID NO:28.
 4. The immunizingcomposition of claim 1, wherein said peptide comprises SEQ ID NO:34. 5.The immunizing composition of claim 1, wherein said peptide is less than35 residues in length.
 6. The immunizing composition of claim 1, whereinsaid peptide is less than 30 residues in length.
 7. The immunizingcomposition of claim 1, wherein said peptide is less than 25 residues inlength.
 8. The immunizing composition of claim 1, wherein said peptideis less than 20 residues in length.
 9. The immunizing composition ofclaim 1, wherein said peptide consists of the sequence of SEQ ID NO:27.10. The immunizing composition of claim 1, wherein said peptide consistsof the sequence of SEQ ID NO:28.
 11. The immunizing composition of claim1, wherein said peptide consists of the sequence of SEQ ID NO:34.